CN115417867A

CN115417867A - Substituted naphthalimide derivative and medical application thereof

Info

Publication number: CN115417867A
Application number: CN202210996213.9A
Authority: CN
Inventors: 郑志兵; 李鹏运; 李松; 肖军海; 钟武; 李行舟; 周辛波; 刘彦东; 樊士勇; 肖典
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2022-12-02

Abstract

The present invention relates to substituted naphthoamide derivatives of formula I or pharmaceutically acceptable salts or hydrates thereof, pharmaceutical compositions comprising said compounds, and methods of use thereofProtein kinase activity, and regulate intracellular and intercellular signaling and responses. The invention also relates to a preparation method of a medicinal composition containing the compound or the medicinal salt or hydrate thereof or the compound and application of the medicinal composition in treating diseases related to neovascularization.

Description

Substituted naphthoyl amide derivative and medical application thereof

Technical Field

The invention relates to substituted naphthamide derivatives, medicinal salts or hydrates thereof and medicinal application of medicinal compositions containing the same. In particular as medicaments for the treatment and/or prophylaxis of diseases which are associated with the tyrosine kinase/tyrosine kinase receptor pathway.

Background

Protein Tyrosine Kinase (TPK) is an important factor in the process of cell signal transmission, participates in a series of cell functions, is closely related to cell growth, differentiation and proliferation, and catalyzes transfer of gamma phosphate of ATP to Tyrosine residues of a plurality of important proteins to phosphorylate phenolic hydroxyl groups, thereby transmitting signals. TPK belongs to the family of protein kinases and can be divided into two broad classes according to their structure: receptor tyrosine protein kinases (RTKs) and Non-Receptor tyrosine protein kinases (Non-Receptor PTKs), which can be further classified into multiple enzyme genera according to structural homology. There are 518 kinase genes in humans, 90 PTKs of which have been discovered, including 58 tyrosine kinases of the receptor type and 32 tyrosine kinases of the non-receptor type. Receptor tyrosine kinases include platelet-derived growth factor receptor (PDGFR), epidermal Growth Factor Receptor (EGFR), fibroblast Growth Factor Receptor (FGFR) and the like, which generally have an extracellular domain, a transmembrane region and an intracellular kinase domain, RTKs are connected inside and outside cells through transmembrane-structured enzyme protein receptors, and activate signal transduction after binding with growth factor ligands. Studies have shown that these receptors and their ligands are important in many tumors, and that many cancers exhibit overexpression of various growth factors, resulting in excessive tyrosine phosphorylation signaling into cells. After being activated, the receptor in the tumor cell is combined with downstream molecules and activated, so that the receptor has tyrosine kinase activity and promotes cell proliferation, and therefore, the activity of RTK in the tumor cell is inhibited, and the receptor-ligand signal transduction is blocked, so that the tumorigenesis and development are effectively inhibited. Non-receptor tyrosine kinases (nrPTKs) generally have no extracellular structure and are usually coupled to cell membranes or exist in the cytoplasm and include members of the Abl kinases, src kinases, C-terminal Src kinases, and the like. nrPTKs are often activated in tumor tissues, and then downstream signal transduction pathways are activated, so that cell proliferation is promoted, apoptosis is resisted, and tumor occurrence and development are promoted.

Angiogenesis is the process by which blood vessels form new sprouts or split off from pre-existing blood vessels. Under normal physiological conditions, angiogenesis is a complex process of coordinated actions of pro-and anti-angiogenic factors. Abnormalities in angiogenesis can cause a range of diseases including retinopathy, arthritis, endometriosis, atherosclerosis and cancer. Folkman has demonstrated in the early 70 s that primary tumor growth, metastasis must be dependent on the formation of new blood vessels. In fact, tumors require new capillaries to provide nutrients and remove metabolic waste products for growth, and the new blood vessels also help in later tumor cell metastasis.

Angiogenesis is tightly controlled and regulated by a number of endogenous pro-and anti-angiogenic factors. The angiogenesis promoting factors include Vascular Endothelial Growth Factor (VEGF), fibroblast Growth Factor (FGF), platelet Derived Growth Factor (PDGF), angiogenin (Tie), ephrin (Ephrin), apelin peptide (apelin/APLN), chemokines, and the like. These factors are often expressed simultaneously and cooperate effectively at different stages of tumor angiogenesis. Among them, vascular endothelial growth factor and its receptor (VEGF/VEGFR) are the most studied and most specific angiogenesis promoting regulatory factors, and can induce endothelial cell differentiation and angiogenesis. VEGF acts on endothelial cells directly to promote mitogenesis and new blood vessel growth, most malignant tumor cells have the function of autocrine VEGF, and the transferred tumor cells release VEGF to stimulate local angiogenesis. VEGFR, a specific receptor for VEGF, is highly expressed in tumor neovasculature. The vascular endothelial cells have genetic stability, so that the VEGFR inhibitor is not easy to generate drug resistance, is easy to reach a target spot and is gathered in a high concentration in tumor tissues. VEGFR is mainly classified into 3 types: VEGFR-1 (FLT-1); VEGFR-2 (KDR) and VEGFR-3 (FLT-4). VEGFR is a highly specific transmembrane receptor, and the protein structure is composed of extracellular, transmembrane and intracellular domain 3 parts. The extracellular region is the binding site of VEGF, and contains 7 immunoglobulin-like functional regions; tyrosine protein kinase (PTK) with 1 insert in the intracellular region; the biological function of VEGFR is to induce division, proliferation and migration of vascular endothelial cells, enhance capillary permeability, extravasate plasma and promote the formation of large quantities of peripheral blood vessels by gradually conducting and amplifying signals through cascade phosphorylation reactions after binding with its ligand to induce the corresponding biological effects of cells. Studies have shown that the VEGF/VEGFR signaling pathway is closely related to tumor angiogenesis and lymphangiogenesis. The signal channel creates a favorable microenvironment for the survival and development of the signal channel, can promote the generation of the microvasculature of the signal channel, obtains rich nutrition supply, and plays a more important role in the tumor diffusion process by the generation of the lymphatic vessels, so that the blocking of the VEGF/VEGFR signal channel can effectively inhibit the generation of the tumor angiogenesis and the lymphatic vessels, and inhibit the growth and the metastasis of the tumor.

Disclosure of Invention

The invention aims to find and invent a novel small molecular compound acting on VEGFR, which can block VEGF/VEGFR related signal channels and has strong angiogenesis inhibition effect, thereby having anti-tumor activity and improving, preventing and treating other various diseases accompanied with abnormal proliferation of neovascularization.

The inventor finds that the compound with the following general formula I can act on a VEGF/VEGFR signal channel, obviously inhibit the activity of protein kinases such as VEGF and the like, and has the effects of inhibiting the generation of new blood vessels and resisting tumors.

The present invention relates in a first aspect to a compound of formula I:

wherein, R is an unsubstituted five-membered heterocyclic ring, and the five-membered heterocyclic ring comprises 1 to 4 heteroatoms selected from O, S, N and Se.

A second aspect of the present invention relates to a pharmaceutical composition comprising at least one compound of formula I or a pharmaceutically acceptable salt thereof or a hydrate thereof as a carrier or excipient.

A third aspect of the present invention relates to the use of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, for the treatment and/or prevention of diseases associated with the tyrosine kinase/tyrosine kinase receptor pathway, including but not limited to: atherosclerosis or pulmonary fibrosis, retinopathy, endometriosis, arthritis, cancer and the like.

A fourth aspect of the present invention is directed to methods for treating and/or preventing diseases associated with the VEGF/VEGFR pathway comprising administering to a patient in need of treatment and/or prevention of diseases associated with the VEGF/VEGFR pathway, including but not limited to: atherosclerosis or pulmonary fibrosis, retinopathy, endometriosis, arthritis, cancer and the like.

A fifth aspect of the present invention relates to a method for the treatment and/or prevention of cancer diseases associated with the VEGF/VEGFR pathway comprising administering a prophylactically and/or therapeutically effective amount of at least one compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, to a patient in need of treatment and/or prevention of cancer diseases associated with the VEGF/VEGFR pathway. Such cancer diseases include, but are not limited to: one or more of gastric cancer, oral cancer, esophageal cancer, thyroid cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, carcinoma of large intestine, renal cancer, osteosarcoma, medulloblastoma, rhabdomyosarcoma and glioblastoma.

According to the invention, the compounds of formula (I) or pharmaceutically acceptable salts or hydrates thereof according to the invention are preferably the following compounds:

according to the present invention, the compounds of the present invention can be prepared by the following reaction scheme:

taking 2-methoxy-4-methyl aminobenzoate (1) as an initial raw material, firstly reacting the raw material 1 with 2,2-dimethyl-1,3-dioxane-4,6-diketone (micellic acid) to prepare 4- [ (2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-methylene) amino ] -2-methoxybenzoic acid methyl ester (3), cyclizing 2 under the high-temperature condition mediated by diphenyl ether to prepare 7-methoxy-4-oxo-1,4-dihydroquinoline-6-carboxylic acid methyl ester (4), chlorinating the 3 by thionyl chloride to prepare 4-chloro-7-methoxyquinoline-6-carboxylic acid methyl ester (5), and obtaining a key intermediate 6 by ammonolysis reaction of the 4. 1-naphthoic acid (7) is used as a raw material to prepare an intermediate 10 through methanesulfonic acid and alkali fusion. In DMF medium, hobt, EDCI is used as condensing agent, and the intermediate reacts with various amines to synthesize the target compound 12.

According to the present invention, the term "pharmaceutically acceptable salts" of the compounds of the present invention includes acid salts of the compounds of the present invention with pharmaceutically acceptable inorganic or organic acids or base salts with pharmaceutically acceptable bases. Wherein acid salts include, but are not limited to: hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogen phosphate, acetate, propionate, butyrate, oxalate, pivalate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate; base salts include, but are not limited to: ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, organic base salts such as dicyclohexylamine and N-methyl-D-glucamine salts, and amino acid salts such as arginine and lysine salts.

According to the present invention, the term base for the compound of the present invention is not particularly limited, as long as it is generally known as a base in organic synthesis, and examples thereof include sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydride, potassium hydride, sodium tert-butoxide, potassium tert-butoxide, cesium carbonate, triethylamine, trimethylamine, N-methylmorpholine, N, N-dimethylaniline, pyridine, isoquinoline, potassium hydroxide, sodium methoxide, potassium methoxide and the like.

Cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

When administered topically to the lower intestinal tract, the compounds of the present invention may be formulated in the form of rectal suppositories or suitable enemas as described above, or alternatively, topical transdermal patches may be used.

The compounds of the present invention may also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oleaginous suspensions, or sterile injectable solutions, in which case the carriers and solvents that may be employed include water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

It is further noted that the specific dosage and method of administration of the compounds of the present invention for each individual patient will depend upon a variety of factors including the age, body weight, sex, physical condition, nutritional status, the activity level of the compound, the time of administration, the metabolic rate, the severity of the condition, and the subjective judgment of the treating physician.

The specific implementation mode is as follows:

the following examples are illustrative of preferred embodiments of the present invention and are not intended to limit the invention in any way.

For all of the following examples, standard procedures and purification methods known to those skilled in the art may be used. Unless otherwise indicated, all temperatures are expressed in degrees Celsius. The structure of the compounds was confirmed by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). The melting point of the compounds was determined by a model RY-1 melting point apparatus, the thermometer being uncorrected and given in degrees Celsius. ¹ HNMR was measured by a Japanese electronic JNM-ECA-400 type nuclear magnetic resonance apparatus. Mass spectra were determined by an API3000 (ESI) type mass spectrometer. All solvents for the reaction are not indicated to have been subjected to a standardized pretreatment.

In the following examples,% means mass% unless otherwise specified.

Example 1: synthesis of 4- [5- [ (thiazolyl) carbamoyl) ] naphthalen-2-oxy ] -7-methoxy-6-quinolinecarboxamide (LYD-2-45)

1.1 4- [ (2,2-dimethyl-4,6-dioxo-1,3-dioxane-5-methylene) amino ] -2-methoxybenzoic acid methyl ester (3)

50.0g (347.0 mmol) of cyclopropanedicarboxylic acid cycloisopropyl ester, 100.0g (942.0 mmol) of trimethyl orthoformate and 125mL of isopropanol were put in a 250mL three-necked flask, and stirred under reflux for 40min. 25g (138.0 mmol) of methyl 2-methoxy-4-aminobenzoate are added and the mixture is stirred under reflux for 10min. Cooled to room temperature, filtered, and the filter cake is washed with ether (50 mL. Times.2) and dried in vacuo to yield 42.75g of yellow crystalline solid, 92.4% yield, mp.200-202 ℃. ¹ H-NMR(400MHz， DMSO-d ₆ )δppm：11.22(s，1H)，8.67(s，1H)，7.69(d，J＝8.5Hz，1H)，7.40(d，J＝2.0Hz，1H)， 7.16(dd，J＝8.5，2.1Hz，1H)，3.84(s，3H)，3.76(d，J＝16.9Hz，3H)，1.67(d，J＝17.1Hz，6H)。 ESI-MSm/z：336.1[M+H] ⁺ 。

1.2 7-methoxy-4-oxo-1,4-dihydroquinoline-6-carboxylic acid methyl ester (4)

20.0g (59.6 mmol) of the product (3) 1.1 and 100mL diphenyl ether are added into a 250mL round-bottom flask, heated to 200 ℃ and stirred for 30min, cooled to 40 ℃ after the reaction is finished, added with ether and stirred for 30min. Filtering, washing the filter cake with ether, vacuum drying to obtain 12.6g of light yellow solid (4), yield 90.5%, mp.243-246 ℃. ¹ H-NMR(400MHz， DMSO-d ₆ )δppm：11.64(s，1H)，8.39(s，1H)，7.83(dd，J＝7.5，5.8Hz，1H)，6.98(s，1H)，5.95(dd，

According to the present invention, the pharmaceutical compositions of the present invention comprise an effective amount of a compound of formula (I) of the present invention or a pharmaceutically acceptable salt or hydrate thereof and one or more suitable pharmaceutically acceptable carriers, where pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, polyethylene-polyoxypropylene-block polymers and lanolin.

According to the present invention, a pharmaceutical composition of a compound of the present invention may be administered in any of the following ways: oral, aerosol inhalation, rectal, nasal, buccal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or via an external reservoir. Of these, oral, intraperitoneal or intravenous administration is preferred. In addition, for the compounds of the present invention to be effective in treating central nervous system disorders, intraventricular administration may be preferred to overcome the potentially low blood brain barrier permeability of the compounds.

When administered orally, the compounds of the present invention may be formulated into any orally acceptable dosage form including, but not limited to, tablets, capsules, aqueous solutions or suspensions. Among them, the carriers generally used for tablets include lactose and corn starch, and additionally, a lubricant such as magnesium stearate may be added. Typical diluents used in capsule formulations include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. If desired, sweetening, flavoring or coloring agents may be added to the above oral dosage forms.

When administered rectally, the compounds of the present invention are generally prepared in the form of suppositories by mixing the drug with a suitable non-irritating excipient. The excipients are in a solid state at room temperature and melt to release the drug at rectal temperature.

When the compound is used for local administration, particularly for treating affected surfaces or organs which are easy to reach by local external application, such as eyes, skin or lower intestinal tract neurogenic diseases, the compound can be prepared into different forms of local administration preparations according to different affected surfaces or organs, and the specific description is as follows:

when topically applied to the eye, the compounds of the present invention may be formulated as a micronized suspension or solution in a carrier such as isotonic sterile saline at pH, with or without the addition of preservatives such as benzylalkenoxide chloride.

When applied topically to the skin, the compounds of the present invention may be formulated in a suitable ointment, lotion or cream formulation in which the active ingredient is suspended or dissolved in one or more carriers. Mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, j =7.5,1.1hz, 1h), 3.85 (s, 3H), 3.77 (s, 3H). ESI-MSm/z:234.2[ M ] +H] ⁺ 。

1.3 4-chloro-7-methoxyquinoline-6-carboxylic acid methyl ester (5)

5.0g (21 mmol) of 1.2 product (4), 15mL of thionyl chloride were added to a 100mL round bottom flask and stirred at reflux for 1h under nitrogen. The thionyl chloride was evaporated under reduced pressure and the remaining pale yellow solid obtained was added to 200mL of saturated sodium bicarbonate solution (containing 3mL of ethyl acetate), stirred until no air bubbles emerged, filtered, and the filter cake was washed with water and used directly for the next reaction.

1.4 4-chloro-7-methoxyquinoline-6-carboxamide (6)

The filter cake of 1.3 (intermediate 5) was then divided into 150mL portionsSeveral 28% ammonia, 5mL ethyl acetate were added to the round bottom flask and stirred at room temperature for 6h. Filtering, washing filter cake with water, vacuum drying to obtain light yellow solid (5) 3.86g, yield 76.1%, mp.214-218 deg.c. ¹ H-NMR(400MHz，DMSO-d ₆ )δppm：8.78(d，J＝4.8Hz，1H)，8.46(s，1H)，7.80(s，1H)， 7.89(s，1H)，7.59(s，1H)，7.64(d，J＝4.8Hz，1H)，4.00(s，3H)。ESI-MSm/z：237.0[M+H] ⁺ 。

1.5 6-sodium sulfonate-1-naphthoic acid (9)

5.0g (29.04 mmol) of 1-naphthoic acid (7) was charged into a 50ml eggplant type bottle, and 6.0ml of concentrated sulfuric acid was charged into the eggplant type bottle at normal temperature. Slowly heating to 115 ℃ and keeping the temperature for 3h. TLC monitoring, reaction was complete. The reaction mother liquor was slowly added dropwise to 30ml of water, cooled to room temperature and then adjusted to pH 4 with saturated NaOH (the solution changed from wine-red to milky white with a large amount of solid precipitated). And (3) carrying out suction filtration, washing a filter cake with a small amount of distilled water to obtain a light yellow solid, and carrying out vacuum drying to obtain a product 5.01g with the yield of 62.7%. ¹ H-NMR(400MHz，DMSO-d ₆ )δppm：13.16(s，1H)，8.81(d，J＝9.0Hz，1H)，8.27～8.20(m， 2H)，8.15(dd，J＝7.2，1.2Hz，1H)，7.82(dd，J＝9.0，1.8Hz，1H)，7.64～7.53(m，1H)。 ESI-MSm/z：257[M-H]-。

1.6 6-hydroxy-1-naphthoic acid (10)

5.0g (18.25 mmol) of sodium 6-sulfonate-1-naphthoic acid (8) were ground into a fine powder in a mortar and divided into three batches. 15.0g (165.0 mmol) of KOH are added into a 100ml crucible, the crucible is heated to about 300 ℃ by an electrothermal sleeve (KOH is melted), and the ground 6-sodium sulfonate-1-naphthoic acid is added into the crucible in batches (stirring is carried out while adding in the heating process). After the addition, the mixture was stirred at a constant temperature for 10 minutes, and the heating was stopped. The mixture was cooled to about 50 ℃ and 25.0ml of water was slowly added to make the liquid in the crucible cloudy. The liquid in the crucible was then transferred to a beaker and the pH adjusted to around 2 with concentrated HCl (a large amount of off-white solid precipitated out of solution during adjustment). Suction filtration, filter cake washing with a little water, vacuum drying to get 1.55g of off-white solid, yield 44.9%. ¹ H-NMR(400MHz， DMSO-d ₆ )δppm：13.03(s，1H)，9.93(s，1H)，8.72(d，J＝10.1Hz，1H)，8.02～7.76(m，2H)，7.45(dd， J＝8.1，7.3Hz，1H)，7.30-6.98(m，2H)。ESI-MSm/z：257[M-H] ^- 。

1.7 6- (6-carbamoyl-7-methoxyquinolin-4-yloxy) -1-naphthoic acid (11)

6-hydroxy-1-naphthoic acid (10) (1.08g, 5.74mmol), cesium carbonate (5.2g, 16.02mmol), and DMF (11.0 ml) were sequentially added to an eggplant type flask (protected with nitrogen), and stirred at room temperature for 10min. Then adding 4-chloro-7-methoxyquinoline-6-formamide (5) (0.8g, 3.39mmol) into an eggplant-shaped bottle, stirring at normal temperature for 10min, slowly heating to 75 ℃, reacting at constant temperature for 5h, monitoring by TLC that the reaction is complete, cooling the reaction liquid to room temperature, adding 20.0ml of distilled water, and reacting with saturated NaHCO ₃ The pH is adjusted to 4-5, and a large amount of solid is separated out. And (4) carrying out suction filtration, washing a filter cake with a small amount of water, and carrying out vacuum drying to obtain a white solid. Yield 1.7g, yield: 68.9 percent. ¹ H-NMR(400MHz，DMSO-d ₆ )δppm：9.05(d，J＝9.5Hz，1H)，8.73(s，1H)，8.69(d，J＝5.2Hz， 1H)，8.20(s，1H)，8.18(s，1H)，7.96(d，J＝2.5Hz，1H)，7.90(s，1H)，7.78(s，1H)，7.67(t， J＝5.3Hz，1H)，7.65～7.62(m，1H)，7.56(s，1H)，6.64(d，J＝5.2Hz，1H)，4.05(s，3H)。 ESI-MSm/z：387[M-H] ^- 。

1.9 Synthesis of 4- {5- [ (4-fluorophenyl) carbamoyl) ] naphthalene-2-oxy } -7-methoxy-6-quinolinecarboxamide

0.30g (0.77 mmol) 6- (6-carbamoyl-7-methoxyquinolin-4-yloxy) -1-naphthoic acid (11), 0.30g EDCI (1.54 mmol), 0.21g Hobt (1.54 mmol) and DMF (5.0 ml) were placed in an eggplant type flask under nitrogen protection and stirred under ice bath conditions. The reaction was monitored by TLC after 1h for completion. Then 2-thiazolamine is added into an eggplant type bottle, ice bath is removed after the 2-thiazolamine is added, the mixture is reacted for 5 hours at normal temperature, and the reaction is monitored by TLC to be complete. 50.0ml of distilled water was added to the reaction solution and stirred for 30min, followed by suction filtration, washing the filter cake with distilled water (10.0 ml. Times.3), vacuum drying to give a pale yellow solid, and column chromatography (dichloromethane-methanol, volume ratio 20: 1) to give 0.07g.1H-NMR (400MHz, DMSO-d 6) delta ppm:12.89 (s, 1H), 8.73 (s, 1H), 8.70 (d, J =5.2 hz, 1h), 8.41 (d, J =9.1hz, 1h), 8.16 (d, J =8.5hz, 1h), 7.98 (d, J =2.7hz, 1h), 7.95-7.88 (m, 2H))，7.79(s，1H)，7.76～7.66(m，1H)，7.63(dd，J＝9.3，2.6Hz，1H)，7.59(d，J＝3.6Hz，1H)，7.56 (s，1H)，7.36(d，J＝3.6Hz，1H)，6.64(d，J＝5.2Hz，1H)，4.05(s，3H)。ESI-MS m/z：471.1[M+H] ⁺ ， 493.1[M+Na] ⁺ 。

Example 2:4- {5- [ (isoxazolyl) carbamoyl) ] naphthalene-2-oxy } -7-methoxy-6-quinolinecarboxamide (LYD-2-49)

The procedure is as in example 1, and column chromatography (dichloromethane-methanol, 20: 1 by volume) gives 0.06g. ¹ H-NMR(400MHz，DMSO-d6)δppm：11.74(s，1H)，8.91(d，J＝1.3Hz，1H)，8.74(s，1H)，8.72(d，J＝5.2Hz，1H)， 8.39(d，J＝9.6Hz，1H)，8.14(d，J＝9.5Hz，1H)，7.98(d，J＝2.5Hz，1H)，7.90(s，1H)，7.86(dd，J ＝7.1，0.9Hz，1H)，7.78(s，1H)，7.72～7.65(m，1H)，7.61(dd，J＝8.6，2.1Hz，1H)，7.57(s，1H)， 7.15(s，1H)，6.66(d，J＝5.5Hz，1H)，4.06(s，3H)。ESI-MS m/z：455.1[M+H] ⁺ ，477.1[M+Na] ⁺ 。

Example 3: inhibition of VEGFR-2 kinase activity by compounds

1) Preparation of stock solutions of compounds

The compounds LYD-2-45 and LYD-2-49 were dissolved in DMSO to prepare stock solutions of appropriate concentrations. The compounds used were stored in a desiccator at room temperature for three months, others could be stored at-20 ℃ for a long period of time.

2) Preparation of working fluid

Compounds were diluted in DMSO, 3-fold gradient diluted, 12 concentration points. The initial concentration is determined by the initial sieving activity of the compound, 0.05mM, or 1mM and so on.

3) Preparation of 1x kinase buffer

1 volume of 5X enzymatic buffer plus 4 volumes of distilled water; 5mM MgCl ₂ ；1mM DTT；1mM MnCl ₂ ；

4) Titration of the kinase VEGFR-2 (100. Mu.M ATP,1 uMTK-substrate-biotin)

5ng/uL of 5XVEGFR-2 was prepared using 1 Xkinase buffer.

5ng/uL of 5XVEGFR-2 was diluted 2-fold with 1x kinase buffer, 8 concentration points +0.

VEGFR-2, step b, was diluted in a gradient, was added to 384 experimental plates (784075, greiner), 2ul/well. Each well was centrifuged for 30s at 1000g with 4ul 1x kinase buffer. 5uM of 5X TK-substrate-biotin and 500. Mu.M of 5X ATP were prepared using 1X kinase buffer.

384 plates were plated with 2. Mu.L TK-substrate-biotin and 2ul ATP per well. The final starting concentration of the kinase VEGFR-2 was 1ng/uL; TK-substrate-biotin and ATP concentrations were 1uM and 100. Mu.M, respectively. Centrifuge at 1000g for 30s. Sealing the plate, and standing at room temperature for 40min. 250nM 4X Sa-XL665 were prepared using HTRF detection buffer.

384 shifts were dosed with 5. Mu.L of Sa-XL665 and 5ul of TK-antibody-Cryptate prepared in step i per well. Centrifuge at 1000g for 30s, and leave at room temperature for 60min.

Envision 2104 plate reader reads fluorescence values. Excitation light: 320nm; light emission: 620nm (Cryptote) and 665nm (XL 665).

5) Determination of the Km value of ATP

5xVEGFR-2 was prepared at 0.156ng/uL using 1 xkinase buffer. Add 2. Mu.L of 5X VEGFR-2 to each well of 384 experimental plates.

In 384 plates, 4ul 1x kinase buffer was added to each well and centrifuged at 1000g for 30s. 5uM of 5X TK-substrate-biotin was prepared using 1X kinase buffer. ATP was diluted with a 3-fold gradient of 1x kinase buffer, starting at 300uM,12 spots +0.

mu.L of TK-substrate-biotin (see step d) and 2ul of ATP (see step e) were added to each well of 384 assay plates. Centrifuge at 1000g for 30s. Sealing the plate, and standing at room temperature for 40min. 250nM 4X Sa-XL665 were prepared using HTRF detection buffer.

mu.L of Sa-XL665 (see step i) and 5ul of TK-antibody-Cryptate were added to each well of 384 plates. Centrifuge at 1000g for 30s, and stand at room temperature for 60min.

6) Screening of Compounds

10nl of the compound dilution prepared in the above step was transferred to 384 experimental plates (784075, greiner) with Echo 550. Centrifuge at 1000g for 1min. And (7) closing the plate. 0.156ng/uL of 5x VEGFR-2 was prepared using a 1x kinase buffer. Add step d diluted VEGFR-2 to the 384 well plates in step c above, 2ul/well. Centrifuge at 1000g for 30s, and leave at room temperature for 10min. 5uM of 5 xTK-substrate-biotin and 6. Mu.M of 5 xATP were prepared using 1X kinase buffer. TK-substrate-biotin and ATP were added to the 384 well plates of step f, 2ul per well. Centrifuge at 1000g for 30s. Sealing the plate, and standing at room temperature for 40min.

250nM 4X Sa-XL665 were prepared using HTRF detection buffer.

384 plates were plated with 5. Mu.L of Sa-XL665 (see step k) and 5ul of TK-antibody-Cryptate per well. Centrifuge at 1000g for 30s, and leave at room temperature for 60min.

7) Data analysis

A. Calculate the ratio of 665/620 per hole

B. Plotting and calculating the IC50 of the Compounds

The relationship between% Inhibition and log of compound concentrations was fitted by the method of nonlinear regression (dose-variable slope) using Graphpad 5.0.

Y＝Bottom+(Top-Bottom)/(1+10^((LogIC50-X)*Hill Slope))

X：log of inhibitor concentration

Y：％Inhibition

8) Test results

TABLE 1 IC inhibition of VEGFR-2 kinase by test Compounds ₅₀

Note: compound No. is example no.

Example 4: inhibitory effect of LYD-2-45 and LYD-2-49 on other tyrosine kinases

The inhibitory activity of LYD-245 and LYD-2-49 (see example 3) on other tyrosine kinases including Kit, FGFR1, FGFR2, FGFR3, flt1, PDGF β, met, EGFR, flt4, PDGF α, ret was determined as in example 3. The test results are shown in table 2.

Experimental results show that the LYD-2-45 and LYD-2-49 have excellent inhibitory activity on various tyrosine kinases, and the inhibitory activity of LYD-2-45 and LYD-2-49 in tyrosine kinases such as Kit, FGFR1, FGFR2, FGFR3, flt1, PDGF beta, flt4, PDGF alpha, ret and the like is superior to that of positive drug Lenvatinib.

TABLE 2 IC inhibition of tyrosine kinases such as Kit, FGFR1 by test compounds ₅₀ (μM)

Example 5: LYD-2-45 and LYD-2-49 inhibiting effect on lung cancer A549 nude mouse xenograft tumor growth

The cell model in the embodiment selects an A549 lung cancer cell strain, and the inhibition effect of LYD-2-45 and LYD-2-49 on the growth of the transplanted tumor of the nude mouse tumor cell is determined. The evaluation methods and results are described below.

1. Experimental materials and instruments

1.1 Experimental consumables for bioactivity evaluation

2. Experimental procedure

Pancreatin digests cells in logarithmic growth phase, counts the cells, then suspends the cells in PBS, respectively inoculates 5 × 106A 549 cells at the breast pad of 4-6 week-old female BALB/c nude mice (positions with abundant blood vessels such as axilla or groin are generally selected), each nude mouse injects 0.1mL cell suspension, and each group can be set to 7 according to different experimental requirements. The animal experiment dosing schedule is: solvent control, lenvatinib (30 mg/kg), LYD-2-45 (30 mg/kg), LYD-2-49 (30 mg/kg), was administered orally once daily.

Observing and measuring the size of the tumor growth every 2 days from the time of inoculation, measuring the maximum diameter (L) and the minimum diameter (D) of the tumor,the formula for calculating the size of the tumor volume is V = L × D ² X 1/2. The body weight of nude mice was weighed every 2 days, and after 24 hours from the last administration, the tumor inhibition rate TGI (%) = (Vc-Vt)/(Vc-V) was calculated from the size of the tumor volume ₀ ) *100.Vc, vt and V ₀ Mice were sacrificed for initial tumor volume administered, tumor volume administered group and tumor volume controlled group, and subcutaneously transplanted tumors were weighed for dissection.

3. Test results

As shown in attached figure 1 and table 3, experimental results show that LYD-2-45 and LYD-2-49 significantly inhibit the growth of A549 transplantable tumors, the effect is superior to that of positive drug Lenvatinib under the same dosage, no weight reduction and other toxic and side effects of nude mice occur in the experimental process, and the weight reduction of the nude mice occurs in the Lenvatinib group, which shows that LYD-2-45 and LYD-2-49 have good tolerance.

TABLE 3 inhibitory Effect of LYD-2-45 and LYD-2-49 on A549 nude mouse xenograft tumor proteins

Example 6: LYD-2-45 and LYD-2-49 have effects in inhibiting growth of thyroid cancer 8305C nude mouse xenograft tumor

In the embodiment, the cell model selects thyroid cancer 8305C cell strains, and the inhibition effect of LYD-2-45 and LYD-2-49 on the growth of the transplanted tumor of the nude mouse with tumor cells is measured. The evaluation methods and results are described below.

1. Experimental materials and instruments

1.1 Experimental consumables for bioactivity evaluation

2. Experimental procedure

The procedure is as in example 5.

3. Test results

As shown in attached figure 2 and table 4, experimental results show that LYD-2-45 and LYD-2-49 significantly inhibit the growth of 8305C transplanted tumors, and the effect is superior to that of a positive drug Lenvatinib under the same dosage.

TABLE 4 inhibitory Effect of LYD-2-45, LYD-2-49 on 8305C nude mouse xenografts

Example 7: LYD-2-45 and LYD-2-49 have effect in inhibiting growth of xenograft tumor of liver cancer HepG2 nude mouse

In the embodiment, the cell model selects the liver cancer HepG2 cell strain, and the inhibitory action of LYD-2-45 and LYD-2-49 on the growth of the transplanted tumor of the nude mouse with tumor cells is measured. The evaluation methods and results are described below.

1. Experimental materials and instruments

1.1 Experimental consumables for bioactivity evaluation

2. Experimental procedure

The procedure is as in example 5.

3. Test results

As shown in the attached figure 3 and a table 5, the experimental results show that LYD-2-45 and LYD-2-49 can obviously inhibit the growth of liver cancer HepG2 transplanted tumor, the effect is superior to that of positive drug Lenvatinib under the same dosage, and no weight reduction and other toxic and side effects of nude mice occur in the experimental process, which indicates that LYD-2-45 and LYD-2-49 have good tolerance.

TABLE 5 inhibitory Effect of LYD-2-45 and LYD-2-49 on HepG2 nude mouse xenograft tumor proteins of hepatocarcinoma

Example 8: LYD-2-45 and LYD-2-49 have effect in inhibiting growth of xenograft tumor of nude mouse with hepatocarcinoma SMMC-7721

The cell model in the embodiment selects the liver cancer SMMC-7721 cell strain, and the inhibitory action of LYD-2-45 and LYD-2-49 on the growth of the transplanted tumor of the nude mouse with tumor cells is determined. The evaluation methods and results are described below.

1. Experimental materials and instruments

1.1 Experimental consumables for bioactivity evaluation

2. Experimental procedure

The procedure is as in example 5.

3. Test results

As shown in attached figure 4 and table 5, experimental results show that LYD-2-45 and LYD-2-49 significantly inhibit the growth of liver cancer SMMC-7721 transplanted tumors, the effect is superior to that of a positive drug Lenvatinib under the same dosage, no weight reduction and other toxic and side effects of nude mice occur in the experimental process, and the weight reduction of the nude mice occurs in the Lenvatinib group, which shows that LYD-2-45 and LYD-2-49 have good tolerance.

TABLE 5 inhibitory effects of LYD-2-45 and LYD-2-49 on xenograft tumor proteins of nude mice with liver cancer SMMC-7721

Example 9: LYD-2-45 and LYD-2-49 for inhibiting growth of renal carcinoma 786-O nude mouse xenograft tumor

The cell model in this example selects renal carcinoma 786-O cell line, and measures the inhibitory effect of LYD-2-45 and LYD-2-49 on the growth of tumor transplanted in nude mice. The evaluation methods and results are described below.

1. Experimental materials and instruments

1.1 Experimental consumables for bioactivity evaluation

2. Experimental procedure

The procedure is as in example 5.

3. Test results

As shown in attached figure 5 and table 6, experimental results show that LYD-2-45 and LYD-2-49 can significantly inhibit the growth of renal cancer 786-O transplanted tumors, and the effect is superior to that of the positive drug Lenvatinib under the same dosage.

TABLE 6 inhibitory effects of LYD-2-45 and LYD-2-49 on renal carcinoma 786-O nude mouse xenograft tumor proteins

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Description of the drawings:

FIG. 1 is a diagram showing the effect of LYD-2-45 and LYD-2-49 on the inhibition of xenograft tumor of nude mouse of lung cancer A549

FIG. 2 is a diagram showing the effect of LYD-2-45 and LYD-2-49 on the inhibition of thyroid carcinoma 8305C xenograft tumors in nude mice

FIG. 3 is a diagram showing the effect of LYD-2-45 and LYD-2-49 on the inhibition of xenograft tumor of liver cancer HepG2 nude mouse

FIG. 4 shows the effect of LYD-2-45 and LYD-2-49 on the inhibition of xenograft tumor in nude mice with liver cancer SMMC-7721

FIG. 5 shows the effect of LYD-2-45 and LYD-2-49 on the inhibition of renal carcinoma 786-O xenograft tumors in nude mice.

Claims

1. A compound of formula I, or a hydrate, solvate, pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same.

2. The compound according to claim 1, wherein R in formula I preferably has the structure:

3. the compound of any one of claims 1-2, pharmaceutically acceptable salts, solvates, hydrates thereof, which compound is selected from, but not limited to, the following compounds:

4. a process for the preparation of a compound as claimed in any one of claims 1 to 3.

5. A pharmaceutical composition comprising at least one compound of any one of claim 1, a pharmaceutically acceptable salt, solvate, hydrate thereof, and one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin.

6. Use of a compound according to any one of claims 1 to 3, a pharmaceutically acceptable salt, solvate, hydrate thereof or a pharmaceutical composition according to claim 3, for the manufacture of a medicament for the treatment and/or prevention of diseases associated with the tyrosine kinase/tyrosine kinase receptor pathway. The diseases comprise: atherosclerosis or pulmonary fibrosis, retinopathy, endometriosis, arthritis, cancer and the like.

7. Use of a compound according to any one of claims 1 to 3, a pharmaceutically acceptable salt, solvate, hydrate thereof or a pharmaceutical composition according to claim 3 for the manufacture of a medicament for the treatment and/or prevention of diseases associated with the VEGF/VEGFR pathway. The diseases comprise: atherosclerosis or pulmonary fibrosis, retinopathy, endometriosis, arthritis, cancer and the like.

8. The use according to claims 7 to 8 for the treatment of cancer, wherein the cancer is one or more of gastric cancer, oral cancer, esophageal cancer, thyroid cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer, colorectal cancer, renal cancer, osteosarcoma, medulloblastoma, rhabdomyosarcoma, and glioblastoma.

9. A medicament as claimed in any one of claims 5 to 8, and which may be administered by a suitable route. Such as oral, sublingual, rectal, parenteral, injection (intradermal, subcutaneous, intramuscular, intravenous, intraarterial), pulmonary, nasal, lingual, buccal, dermal, mucosal, conjunctival, topical or in the form of an implant.